Enhancement of the luteinising hormone surge by male olfactory signals is associated with anteroventral periventricular Kiss1 cell activation in female rats.

Olfactory stimuli play an important role in regulating reproductive functions in mammals. The present study investigated the effect of olfactory signals derived from male rats on kisspeptin neuronal activity and luteinising hormone (LH) secretion in female rats. Wistar-Imamichi strain female rats were ovariectomised (OVX) and implanted with preovulatory levels of 17ß-oestradiol (E2 ). OVX+E2 rats were killed 1 hour after exposure to either: clean bedding, female-soiled bedding or male-soiled bedding. Dual staining for Kiss1 mRNA in situ hybridisation and c-Fos immunohistochemistry revealed that the numbers of Kiss1-expressing cells and c-Fos-immunopositive Kiss1-expressing cells in the anteroventral periventricular nucleus (AVPV) were significantly higher in OVX+E2 rats exposed to male-soiled bedding than those of the other groups. No significant difference was found with respect to the number of c-Fos-immunopositive Kiss1-expressing cells in the arcuate nucleus and c-Fos-immunopositive Gnrh1-expressing cells between the groups. The number of c-Fos-immunopositive cells was also significantly higher in the limbic system consisting of several nuclei, such as the bed nucleus of the stria terminalis, the cortical amygdala and the medial amygdala, in OVX+E2 rats exposed to male-soiled bedding than the other groups. OVX+E2 rats exposed to male-soiled bedding showed apparent LH surges, and the peak of the LH surge and area under the curve of LH concentrations in the OVX+E2 group were significantly higher than those of the other two groups. These results suggest that olfactory signals derived from male rats activate AVPV kisspeptin neurones, likely via the limbic system, resulting in enhancement of the peak of the LH surge in female rats. Taken together, the results of the present study suggests that AVPV kisspeptin neurones are a target of olfactory signals to modulate LH release in female rats.

Evidence of involvement of neurone-glia/neurone-neurone communications via gap junctions in synchronised activity of KNDy neurones.

Pulsatile secretion of gonadotrophin-releasing hormone (GnRH)/luteinising hormone is indispensable for the onset of puberty and reproductive activities at adulthood in mammalian species. A cohort of neurones expressing three neuropeptides, namely kisspeptin, encoded by the Kiss1 gene, neurokinin B (NKB) and dynorphin A, localised in the hypothalamic arcuate nucleus (ARC), so-called KNDy neurones, comprises a putative intrinsic source of the GnRH pulse generator. Synchronous activity among KNDy neurones is considered to be required for pulsatile GnRH secretion. It has been reported that gap junctions play a key role in synchronising electrical activity in the central nervous system. Thus, we hypothesised that gap junctions are involved in the synchronised activities of KNDy neurones, which is induced by NKB-NK3R signalling. We determined the role of NKB-NK3R signalling in Ca2+ oscillation (an indicator of neuronal activities) of KNDy neurones and its synchronisation mechanism among KNDy neurones. Senktide, a selective agonist for NK3R, increased the frequency of Ca2+ oscillations in cultured Kiss1-GFP cells collected from the mediobasal hypothalamus of the foetal Kiss1-green fluorescent protein (GFP) mice. The senktide-induced Ca2+ oscillations were synchronised in the Kiss1-GFP and neighbouring glial cells. Confocal microscopy analysis of these cells, which have shown synchronised Ca2+ oscillations, revealed close contacts between Kiss1-GFP cells, as well as between Kiss1-GFP cells and glial cells. Dye coupling experiments suggest cell-to-cell communication through gap junctions between Kiss1-GFP cells and neighbouring glial cells. Connexin-26 and -37 mRNA were found in isolated ARC Kiss1 cells taken from adult female Kiss1-GFP transgenic mice. Furthermore, 18ß-glycyrrhetinic acids and mefloquine, which are gap junction inhibitors, attenuated senktide-induced Ca2+ oscillations in Kiss1-GFP cells. Taken together, these results suggest that NKB-NK3R signalling enhances synchronised activities among neighbouring KNDy neurones, and that both neurone-neurone and neurone-glia communications via gap junctions possibly contribute to synchronised activities among KNDy neurones.

Neonatal Kisspeptin is Steroid-Independently Required for Defeminisation and Peripubertal Kisspeptin-Induced Testosterone is Required for Masculinisation of the Brain: A Behavioural Study Using Kiss1 Knockout Rats.

Rodents show apparent sex differences in their sexual behaviours. The present study used Kiss1 knockout (KO) rats to evaluate the role of kisspeptin in the defeminisation/masculinisation of the brain mechanism that controls sexual behaviours. Castrated adult Kiss1 KO males treated with testosterone showed no male sexual behaviours but demonstrated the oestrogen-induced lordosis behaviours found in wild-type females. The sizes of some of the sexual dimorphic nuclei of Kiss1 KO male rats are similar to those of females. Plasma testosterone levels at embryonic day 18 and postnatal day 0 (PND0) in Kiss1 KO males were high, similar to wild-type males, indicating that perinatal testosterone is secreted in a kisspeptin-independent manner. Long-term exposure to testosterone from peripubertal to adult periods restored mounts and intromissions in KO males, suggesting that kisspeptin-dependent peripubertal testosterone secretion is required to masculinise the brain mechanism. This long-term testosterone treatment failed to abolish lordosis behaviours in KO males, whereas kisspeptin replacement at PND0 reduced lordosis quotients in Kiss1 KO males but not in KO females. These results suggest that kisspeptin itself is required to defeminise behaviour in the perinatal period, in cooperation with testosterone. Oestradiol benzoate treatment at PND0 suppressed lordosis quotients in Kiss1 KO rats, indicating that the mechanisms downstream of oestradiol work properly in the absence of kisspeptin. There was no significant difference in aromatase gene expression in the whole hypothalamus between Kiss1 KO and wild-type male rats at PND0. Taken together, the present study demonstrates that both perinatal kisspeptin and kisspeptin-independent testosterone are required for defeminisation of the brain, whereas kisspeptin-dependent testosterone during peripuberty to adulthood is needed for masculinisation of the brain in male rats.

Numerical study of plasma generation process and internal antenna heat loadings in J-PARC RF negative ion source.

A numerical model of plasma transport and electromagnetic field in the J-PARC (Japan Proton Accelerator Research Complex) radio frequency ion source has been developed to understand the relation between antenna coil heat loadings and plasma production/transport processes. From the calculation, the local plasma density increase is observed in the region close to the antenna coil. Electrons are magnetized by the magnetic field line with absolute magnetic flux density 30-120 Gauss which leads to high local ionization rate. The results suggest that modification of magnetic configuration can be made to reduce plasma heat flux onto the antenna.

Pre-conditioning procedure suitable for internal-RF-antenna of J-PARC RF-driven H⁻ ion source.

The Japan Proton Accelerator Research Complex (J-PARC) cesiated RF-driven H(-) ion source has been successfully operated for about 1 yr. By the world brightest level beam, the J-PARC design beam power of 1 MW was successfully demonstrated. Although no internal-RF-antenna failure, except for the once caused by an excess cesium due to a misoperation, occurred in the operation, many antennas failed in pre-conditionings for the first hundred days. The antenna failure rate was drastically decreased by using an antenna with coating thicker than a standard value and the pre-conditioning procedure repeating 15 min 25 kW RF-power operation and impurity-gas evacuation a few times, before the full power (50 kW) operation.

Fine-tuning to minimize emittances of J-PARC RF-driven H⁻ ion source.

The Japan Proton Accelerator Research Complex (J-PARC) cesiated RF-driven H(-) ion source has been successfully operated for about one year. By the world's brightest level beam, the J-PARC design beam power of 1 MW was successfully demonstrated. In order to minimize the transverse emittances, the rod-filter-field (RFF) was optimized by changing the triple-gap-lengths of each of pairing five piece rod-filter-magnets. The larger emittance degradation seems to be caused by impurity-gases than the RFF. The smaller beam-hole-diameter of the extraction electrode caused the more than expected improvements on not only the emittances but also the peak beam intensity.

Status of the RF-driven H⁻ ion source for J-PARC linac.

For the upgrade of the Japan Proton Accelerator Research Complex linac beam current, a cesiated RF-driven negative hydrogen ion source was installed during the 2014 summer shutdown period, with subsequent operations commencing on September 29, 2014. The ion source has been successfully operating with a beam current and duty factor of 33 mA and 1.25% (500 µs and 25 Hz), respectively. The result of recent beam operation has demonstrated that the ion source is capable of continuous operation for approximately 1100 h. The spark rate at the beam extractor was observed to be at a frequency of less than once a day, which is an acceptable level for user operation. Although an antenna failure occurred during operation on October 26, 2014, no subsequent serious issues have occurred since then.

Lack of pulse and surge modes and glutamatergic stimulation of luteinising hormone release in Kiss1 knockout rats.

Kisspeptin, encoded by the Kiss1 gene, has attracted attention as a key candidate neuropeptide in controlling puberty and reproduction via regulation of gonadotrophin-releasing hormone (GnRH) secretion in mammals. Pioneer studies with Kiss1 or its cognate receptor Gpr54 knockout (KO) mice showed the indispensable role of kisspeptin-GPR54 signalling in the control of animal reproduction, although detailed analyses of gonadotrophin secretion, especially pulsatile and surge-mode of luteinising hormone (LH) secretion, were limited. Thus, in the present study, we have generated Kiss1 KO rats aiming to evaluate a key role of kisspeptin in governing reproduction via pulse and surge modes of GnRH/LH secretion. Kiss1 KO male and female rats showed a complete suppression of pulsatile LH secretion, which is responsible for folliculogenesis and spermatogenesis, and an absence of puberty and atrophic gonads. Kiss1 KO female rats showed no spontaneous LH/follicle-stimulating hormone surge and an oestrogen-induced LH surge, suggesting that the GnRH surge generation system, which is responsible for ovulation, does not function without kisspeptin. Furthermore, challenge of major stimulatory neurotransmitters, such as monosodium glutamate, NMDA and norepinephrine, failed to stimulate LH secretion in Kiss1 KO rats, albeit they stimulated LH release in wild-type controls. Taken together, the results of the present study confirm that kisspeptin plays an indispensable role in generating two modes (pulse and surge) of GnRH/gonadotrophin secretion to regulate puberty onset and normal reproductive performance. In addition, the present study suggests that kisspeptin neurones play a critical role as a hub integrating major stimulatory neural inputs to GnRH neurones, using newly established Kiss1 KO rats, which serve as a useful model for detailed analysis of hormonal profiles.

Dependence of beam emittance on plasma electrode temperature and rf-power, and filter-field tuning with center-gapped rod-filter magnets in J-PARC rf-driven H(-) ion source.

The prototype rf-driven H(-) ion-source with a nickel plated oxygen-free-copper (OFC) plasma chamber, which satisfies the Japan Proton Accelerator Research Complex (J-PARC) 2nd stage requirements of a H(-) ion beam current of 60 mA within normalized emittances of 1.5 π mm mrad both horizontally and vertically, a flat top beam duty factor of 1.25% (500 µs × 25 Hz) and a life-time of more than 50 days, was reported at the 3rd international symposium on negative ions, beams, and sources (NIBS2012). The experimental results of the J-PARC ion source with a plasma chamber made of stainless-steel, instead of nickel plated OFC used in the prototype source, are presented in this paper. By comparing these two sources, the following two important results were acquired. One was that the about 20% lower emittance was produced by the rather low plasma electrode (PE) temperature (TPE) of about 120 °C compared with the typically used TPE of about 200 °C to maximize the beam current for the plasma with the abundant cesium (Cs). The other was that by using the rod-filter magnets with a gap at each center and tuning the gap-lengths, the filter-field was optimized and the rf-power necessary to produce the J-PARC required H(-) ion beam current was reduced typically 18%. The lower rf-power also decreases the emittances.

Operation and development status of the J-PARC ion source.

A cesium-free H(-) ion source driven with a LaB6 filament is being operated at the Japan Proton Accelerator Research Complex (J-PARC) without any serious trouble since the restoration from the March 2011 earthquake. The H(-) ion current from the ion source is routinely restricted approximately 19 mA for the lifetime of the filament. In order to increase the beam power at the linac beam operation (January to February 2013), the beam current from the ion source was increased to 22 mA. At this operation, the lifetime of the filament was estimated by the reduction in the filament current. According to the steep reduction in the filament current, the break of the filament was predicted. Although the filament has broken after approximately 10 h from the steep current reduction, the beam operation was restarted approximately 8 h later by the preparation for the exchange of new filament. At the study time for the 3 GeV rapid cycling synchrotron (April 2013), the ion source was operated at approximately 30 mA for 8 days. As a part of the beam current upgrade plan for the J-PARC, the front end test stand consisting of the ion source and the radio frequency quadrupole is under preparation. The RF-driven H(-) ion source developed for the J-PARC 2nd stage requirements will be tested at this test stand.

Development of an H- ion source for Japan Proton Accelerator Research Complex upgrade.

A cesium (Cs) free H(-) ion source driven with a lanthanum hexaboride (LaB(6)) filament was adopted as an ion source for the first stage of the Japan Proton Accelerator Research Complex (J-PARC). At present, the maximum H(-) ion current produced by the ion source is 38 mA, using which J-PARC can produce a proton beam power of 0.6 MW by accelerating it with the 181 MeV linac and the 3 GeV rapid cycling synchrotron. In order to satisfy the beam power of 1 MW required for the second stage of the J-PARC in the near future, we have to increase the ion current to more than 60 mA. Therefore, we have started to develop a Cs-seeded ion source by adding an external Cs-seeding system to a J-PARC test ion source that has a structure similar to that of the J-PARC ion source except for the fact that the plasma chamber is slightly larger. As a result, a H(-) ion current of more than 70 mA was obtained from the ion source using a tungsten filament instead of a LaB(6) filament with a low arc discharge power of 15 kW (100 V, 150 A).

Recent operation status of Japan Proton Accelerator Research Complex H- ion source.

A cesium-free H(-) ion source driven with a LaB(6) filament is being operated at the Japan Proton Accelerator Research Complex for approximately three years without any serious trouble. In the beam commissioning or supply runs, the ion source has been operated in two different modes such as low current mode of 5 mA and high current mode of 30 mA. The total interruption time during the runs due to the ion source failure is approximately 50 h, which correspond to the ion source availability of 99%. After a long-term operation, the surface of the filament and the plasma electrode become discolored with dark partially. The result of surface analysis with field emission scanning electron microscope showed that most of the dark material is formed with boron. At the beam test performed in the interval of the run, we demonstrated that the H(-) current increased by miniaturizing the LaB(6) filament.

A survey of optimal filament shape in a Japan Proton Accelerator Research Complex H- ion source.

Dependence of various shapes of lanthanum hexaboride (LaB(6)) filaments on H(-) ion currents (I_H(-)'s) was examined by using the first Japan Proton Accelerator Research Complex (J-PARC) test ion source. It is almost the same with the J-PARC H(-) ion source (J-PARC-IS) except for the maximum arc current (290 A instead of 400 A). An I_H(-) of 35.2 mA was extracted by using a cylindrical double-spiral LaB(6) filament with a diameter of 29.5 mm and a length of 35.5 mm, which is the same one used in J-PARC-IS. It increased to 43.4 mA with a flat triple-hairpin LaB(6) filament. The I_H(-) is considered to be increased by the enlargement of the high density plasma region near the plasma electrode aperture and the reduction of the LaB(6) filament unemission area located in the high density plasma region.

An innovative high-power constant-current pulsed-arc power-supply for a high-density pulsed-arc-plasma ion-source using a LaB6-filament.

An innovative high-power constant-current (CC) pulsed-arc (PA) power-supply (PS) indispensable for a high-density PA plasma ion-source using a lanthanum hexaboride (LaB(6)) filament was devised by combining a constant-voltage (CV) PA-PS, which is composed of an insulated gate bipolar transistor (IGBT) switch, a CV direct-current (dc) PS and a 270 mF capacitor with a CC-PA-PS, which is composed of an IGBT-switch, a CC-dc-PS and a 400 microH inductor, through the inductor. The hybrid-CC-PA-PS succeeded in producing a flat arc-pulse with a peak power of 56 kW (400 A x 140 V) and a duty factor of more than 1.5% (600 micros x 25 Hz) for Japan Proton Accelerator Research Complex (J-PARC) H(-) ion-source stably. It also succeeded in shortening the 99% rising-time of the arc-pulse-current to about 20 micros and tilting up or down the arc-pulse-current arbitrarily and almost linearly by changing the setting voltage of its CV-dc-PS.

Interesting experimental results in Japan Proton Accelerator Research Complex H- ion-source development (invited).

The following interesting experimental results observed in Japan Proton Accelerator Research Complex (J-PARC) H(-) ion-source developments are reviewed. It was proven that almost all of H(-) ions were produced with surface reactions in cesium (Cs)-free J-PARC H(-) ion-sources. The world's most intense class H(-) ion current of 38 mA in Cs-free ion sources for a high-energy linac was attained by an optimal shape and high temperature of the plasma electrode (PE), usage of a lanthanum hexaboride (LaB(6)) filament, and a newly devised high-power constant-current pulsed-arc power supply indispensable for it. It was also proven that the H(-) ion current could be increased to more than 40 mA by optimizing LaB(6)-filament shape. The surface elemental analysis of the PE after operation with a LaB(6)-filament showed that it was coated by boron (B) 95.5%, lanthanum (La) 2.5%, and oxygen (O) 1.9%. The H(-) ion current decreased by about 20% when a tungsten (W) filament was used instead of a LaB(6)-filament. The H(-) ion current could not be increased by seeding cesium (Cs) if the LaB(6)-filament was used. On the other hand, it was increased to more than 70 mA with much lower arc current of 150 A if Cs was seeded when a W-filament was used.

Development and operation of a Cs-free J-PARC H(-) ion source.

A cesium-free H(-) ion source driven with a LaB(6) filament was developed for the J-PARC. It was operated for the J-PARC linac beam commissioning, which was started on 20 November 2006. Eight runs of 2 or 3 week beam commissioning were done until the end of June 2007. The source was mainly operated with a duty factor of 0.8% (320 micros and 25 Hz) while providing a 5 mA beam typically. Each interval of the runs, precise optimizations, such as the filament position, and so on, are examined. At present, a H(-) beam with a current of 38 mA and a rms normalized emittance of 0.22 pi mm mrad is extracted with a duty factor of 0.8% (320 micros and 25 Hz).

Homer1c interacts with Hippi and protects striatal neurons from apoptosis.

By the two hybrid screening of mouse brain cDNA library, we identified Hppi, a cell death-promoting protein, as a binding partner of postsynaptic scaffold protein Homer1c. Hippi interacted specifically with Homer1c but not with its homologue Homer2. It was reported that Hippi, when complexed with Hip1, induces the apoptosis in striatal neurons and may cause Huntington's disease. We found that this apoptotic effect of Hippi was specific to the striatum and was not observed in hippocampal neurons. Furthermore, the apoptotic effect of Hippi was prevented when Homer1c was co-expressed in cultured striatal neurons. The protective effect of Homer1c was diminished when Hippi binding domain was deleted. These results suggest that Homer1c may play an important role in the mechanisms of neuronal death in the striatum.

Ultrafast photoinduced melting of a spin-Peierls phase in an organic charge-transfer compound, K-tetracyanoquinodimethane.

Ultrafast photoinduced phase transition in a spin-Peierls (SP) system of K-tetracyanoquinodimethane (K-TCNQ) was studied by femtosecond (fs) reflection spectroscopy. Photocarriers destabilize the SP phase, resulting in a decrease in molecular dimerization within 400 fs. Such a melting of the SP phase drives three kinds of coherent oscillations. By comparing the oscillations with the Raman bands activated by the dimerization, we show that the oscillation of 20 cm-1 is due to an LO phonon, and it plays an important role for the stabilization of the SP phase.

The validity of the mangled extremity severity score in the assessment of upper limb injuries.

The Mangled Extremity Severity Score (MESS) may be used to decide whether to perform amputation in patients with injuries involving a limb. A score of 7 points or higher indicates the need for amputation. We have treated three patients with a MESS of 7 points or higher, in two of which the injured limb was salvaged. This scoring system was originally devised to assess injuries to the lower limb. However, a MESS of 7 points as a justification for amputation does not appear appropriate when assessing injuries to the major vessels in the upper limb.

Non-apoptotic neurite degeneration in apoptotic neuronal death: pivotal role of mitochondrial function in neurites.

The length and thinness of neurites render them greatly susceptible to a variety of insults. Accumulating evidence suggests that neurite degeneration is not a passive, but an active and causative, event in some neurodegenerative diseases. Nonetheless, the mechanisms underlying neurite degeneration remain largely unknown. To elucidate the relevant mechanisms, we employed a mutant C57BL/Wld mouse with a unique phenotype of resistance to Wallerian degeneration, and separately analyzed the destruction of cell soma and neurites following treatment with vinblastine, a microtubule-disrupting agent, in superior cervical ganglion neurons. Vinblastine induced macromolecular synthesis-dependent cell death, which was indistinguishable between the wild-type and mutant mice. Evidence for a loss of mitochondrial cytochrome c, caspase activation, and nuclear fragmentation, has indicated that this type of cell death is entirely apoptotic. Consistent with this, the ATP level in the cell soma was well maintained and indistinguishable between wild-type and mutant mice. In neurites of wild-type neurons, vinblastine induced an early loss of mitochondrial membrane potential (MMP) and ATP depletion preceding caspase-independent degeneration, suggesting that this type of neurite degeneration is principally non-apoptotic. In contrast, neurites of mutant neurons were markedly resistant to vinblastine-induced degeneration, and both the MMP and the ATP content in the neurites were well maintained. Exposure of mutant neurons to carbonyl cyanide m-chlorophenyl-hydrazone, an uncoupler, caused extreme neurite degeneration following rapid MMP loss. Collectively, our findings suggest that: 1) neurite degeneration is regulated through a non-apoptotic process achieved by mitochondrial dysfunction in neurites; 2) the mitochondrial functional status is controlled separately in neurites and in the neuronal soma.